solubilization purification

Solubilization,purification and reconstitution of Ca 2+-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents:Preservation of native structure and function of the enzyme by DHPCAmritlal Mandal,Sudip Das,Tapati Chakraborti,Pulak Kar,Biswarup Ghosh,Sajal Chakraborti *Department of Biochemistry and Biophysics,University of Kalyani,Kalyani 741235,West Bengal,India Received 30June 2005;received in revised form 21September 2005;accepted 27September 2005Available online 19October 2005AbstractThe properties of Ca 2+-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)}were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC),poly(oxy-ethylene)8-lauryl ether (C 12E 8)and Triton X-100as the solubilizing agents.Solubilization with DHPC consistently gave higher yields of purified Ca 2+-ATPase with a greater specific activity than solubilization with C 12E 8or Triton X-100.DHPC was determined to be superior to C 12E 8;while that the C 12E 8was determined to be better than Triton X-100in active enzyme yields and specific activity.DHPC solubilized and purified Ca 2+-ATPase retained the E1Ca ÀE1*Ca conformational transition as that observed for native microsomes;whereas the C 12E 8and Triton X-100solubilized preparations did not fully retain this transition.The coupling of Ca 2+transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes,whereas that the coupling was much lower for the C 12E 8and Triton X-100purified enzyme reconstituted in liposomes.The specific activity of Ca 2+-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC)vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C 12E 8and Triton X-100,respectively.Addition of the protonophore,FCCP caused a marked increase in Ca 2+uptake in the reconstituted proteoliposomes compared with the untreated liposomes.Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223nm is well correlated with decreased specific activity.It,therefore,appears that the DHPC purified Ca 2+-ATPase retained more organized and native secondary conformation compared to C 12E 8and Triton X-100solubilized and purified preparations.The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C 12E 8and Triton X-100preparations have to some extent smaller size.These studies suggest that the Ca 2+-ATPase solubilized,purified and reconstituted with DHPC is superior to that obtained with C 12E 8and Triton X-100in many ways,which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.D 2005Elsevier B.V .All rights reserved.Keywords:Ca 2+-ATPase;Endoplasmic reticulum;Bovine pulmonary artery smooth muscle microsome;Liposome;DHPC;C 12E 8;Triton X-100;DOPC;FCCP1.IntroductionChanges in the intracellular Ca 2+concentration [(Ca 2+)i ]regulate the contraction–relaxation cycle of smooth muscle [1].Plasma membrane Ca 2+-ATPase and endoplasmic reticular (ER)Ca 2+-ATPase play important roles in counteracting an increase in [(Ca 2+)i ]in smooth muscle caused by different agonists [1].Recent research provided evidence suggesting that the ER Ca 2+-ATPase is one of the intrinsic Ca 2+transport membrane protein,which is involved in Ca 2+uptake in the suborganelle [2].Three genes of ATP2A1,ATP2A2and ATP2A3encode three types of SERCA protein.SERCA 1,0304-4165/$-see front matter D 2005Elsevier B.V .All rights reserved.doi:10.1016/j.bbagen.2005.09.013Abbreviations:ER,endoplasmic reticulum;SR,sarcoplasmic reticulam;DHPC,1,2-diheptanoyl-sn-phosphatidylcholine;C 12E 8,poly(oxy-ethylene)8-lauryl ether;DOPC,dioleoyl-phosphatidylcholine;DTT,D,L-1,4-dithiothreitol;HBPS,Hank’s buffered physiological saline;MOPS,3-(N -morpholino)propa-nesulphonic acid;TBS,tris-buffered saline;EGTA,ethylene glycol bis(2-aminoethyl ether)-N,N,N V ,N V -tetraacetic acid;PMSF,phenylmethylsulfonyl fluoride;DFBAPTA,5-5V difluoro derivative of 1,2-bis(o-aminophenox-y)ethane-N,N,N V ,N V ,tetraacetic acid;CD,circular dichroism;FCCP,carbonyl-cyanide-p -trifluoromethoxyphenylhydrazone*Corresponding author.Tel.:+919831228224;fax:+913325828282.E-mail address:saj _chakra@ (S.Chakraborti).Biochimica et Biophysica Acta 1760(2006)20–31SERCA2and SERCA3and alternative splicing of the three primary transcripts gives rise to at least nine SERCA isoforms with different functions[3–7].SERCA2b isoform having mol mass of¨115kDa[8]is the principal form of the Ca2+-ATPase in smooth muscles and appears to represent a generic‘‘ER’’form of the Ca2+-ATPase[9].Ca2+-ATPase couples the transport of2mol of Ca2+across the ER membrane with the hydrolysis of1mol of ATP and a conformational change accompanies the reaction cycle[10–12].To elucidate fully the structure and function of Ca2+-ATPase,and particularly the role of lipidÀprotein interactions in the conformational changes that accompany ion transloca-tion,it is necessary to isolate the protein and separate it from other membrane constituents.The most successful approach so far involves the use of detergents for solubilization[12–19] and for reconstitution into defined lipids[20–29].A major challenge in the solubilization and reconstitution of membrane proteins is the choice of a detergent that preserves the native protein structure and biological function.Nonionic detergents such as C12E8and Triton X-100have been employed for the solubilization and purification of Ca2+-ATPase from sarcoplas-mic reticulum(SR)[12,13,16,17].These procedures have produced varied results in terms of active enzyme yield, specific activity and formation of phosphorylated enzyme. Reconstitution of membrane proteins into liposomes provides a powerful tool in structural as well as functional areas of membrane protein research[24,30].It has been shown that in some systems,for example,sarcoplasmic reticulum,the nature of the detergent used for reconstitution is a key factor in determining the properties of the reconstituted system[30].In some membrane preparations,C12E8and Triton X-100have emerged suitable detergents for optimizing the recovery of membrane bound enzymes[18,24,29–35].To the best of our knowledge,reconstitution of pulmonary artery smooth muscle microsomal(enriched with ER)Ca2+-ATPase into defined lipids has not been performed with any study based on these detergents.A short chain phospholipid detergent,DHPC has been described by Kessi et al.[30]and Shivanna and Rowe [35]for solubilizing membrane proteins[35].They showed that DHPC is a better solubilizing agent for a variety of membrane proteins,while retaining their maximal native activity.We have applied DHPC for solubilization,purification and reconstitution of Ca2+-ATPase from bovine pulmonary artery smooth muscle microsomes.In this present communication,we compared the effectiveness in solubilizing and purifying Ca2+-ATPase using DHPC with that prepared by using C12E8and Triton X-100by studying simultaneous measurements of Ca2+-ATPase activity and ATP dependent Ca2+uptake.Furthermore, detailed comparison of the DHPC,C12E8and Triton X-100 purified preparations was made by tryptophan fluorescence and also by circular dichroism spectroscopy.Reconstitution of Ca2+-ATPase into dioleoylphosphatidylcholine(DOPC)using DHPC,C12E8and Triton X-100were performed and the resulting activities were compared.Our results demonstrate that the DHPC purified and reconstituted Ca2+-ATPase into DOPC gives a higher activity compared to that of C12E8and Triton X-100purified preparations.To our knowledge,this is the first report for purification and reconstitution of Ca2+-ATPase of bovine pulmonary artery smooth muscle microsomal Ca2+-ATPase into DOPC using the short-chain phospholipid detergent DHPC.2.Materials and methods2.1.MaterialsDHPC and DOPC were obtained from Avanti Polar Lipids.C12E8,Triton X-100,Tris-ATP,PMSF,sucrose,DTT,CaCl2,rotenone,NADPH,AMP,p-nitrophenylphosphate,p-nitrophenol,cytochrome c,EGTA,Reactive Red120-agarose,FCCP and horseradish peroxidase conjugated secondary antibody were purchased from Sigma Chemical Co.,St.Louis,MO,USA.Sephadex G-100was procured from Pharmacia,Upsala,Sweden.Bio-Beads SM-2(average pore diameter90A˚)was purchased from Bio-Rad,California,USA.DFBAPTA and Fura Red,tetraammonium salt were the products of Molecular Probes,OR, USA.BCA protein assay kit was obtained from Pierce,Rockford,Illinois, USA.Control SERCA2b and its polyclonal antibody were supplied by Dr. Jonathan Lytton,University of Calgary,Calgary,Canada.All other chemicals used were of analytical grade.2.2.Methods2.2.1.Microsome preparationBovine pulmonary artery collected from slaughterhouse was washed several times with Hank’s buffered physiological saline(HBPS)(137mM NaCl, 1.1mM MgCl2, 4.69mM KCl, 3.7mM HEPES buffer,11mM Glucose,pH7.4)and kept at4-C.The washed pulmonary artery was used for further processing within1h after collection.The intimal and serosal (external)layers were removed and the tunica media,i.e.,the smooth muscle tissue was collected and used for the present studies[36].The smooth muscle tissue was characterized by histology with eosin–hematoxylin stain in a light microscope[37].Microsomes enriched with the ER from the smooth muscle tissue were prepared by following the procedure described by Chakraborti et al.[38].2.2.2.Characterization of microsomes by the assay of marker enzymesRotenone-insensitive cytochrome c reductase activity was measured as previously described[39].Cytochrome c oxidase was assayed by the procedure of Wharton and Tzagoloff[40].Acid phosphatase activity was determined at pH5.5using p-nitrophenylphosphate as the substrate[41].Release of Pi from 5V AMP,an index of5V-nucleotidase activity,was determined by the method of Chen et al.[42].2.2.3.Determination of proteinProtein concentration was estimated by the Pierce Micro BCA protein assay kit(Pierce,Rockford,Illinois,USA)by following the procedure of Smith et al.[43]using bovine serum albumin(BSA)as the standard.2.2.4.Solubilization and Purification of Ca2+-ATPase from the smooth muscle microsomeAll buffers used contain1mM PMSF,1A M pepstatin and10A M leupeptin unless stated otherwise.All purification steps were performed at 4-C.2.2.4.1.Solubilization of microsomal vesicles.The freshly prepared microsomes were solubilized on ice with detergents(DHPC,C12E8and Triton X-100)as described by Kessi et al.[30]with some modifications.Briefly,200 mM stock solutions of detergents in solubilization buffer(50mM Tris–HCl,1 M KCl,20%glycerol,1mM DTT,pH7.5)were added to freshly prepared microsomes to the desired final detergent concentration at a constant microsomal vesicle protein concentration of6mg/ml in the same buffer. Overall,the detergent/protein ratio appears to be1:1for Triton X-100;1:0.9forA.Mandal et al./Biochimica et Biophysica Acta1760(2006)20–3121C12E8;and1.5:1for DHPC,which have been determined by us to be optimum for enzyme solubilization and optimum activity.Unless stated otherwise,microsomal vesicles were solubilized at0-C by drop-wise addition of stock detergent solution with vortex-mixing for60s. Solubilization continued on ice for30min with intermittent mixing.Final concentrations of each of the detergents were varied between0and50mM. The solubilization mixture was centrifuged at105,000Âg in an Ultracentrifuge (Beckman,USA)at4-C for25min.The enzyme activity was determined in both the supernatants and the pellets[30].2.2.4.2.Reactive Red120-agarose chromatography.Microsomes were solubilized with the buffer A(50mM Tris–HCl buffer,pH7.5,0.5 mM MgCl2,2mM DTT,20%glycerol,0.5mM EGTA and50A M CaCl2) containing the respective detergents(DHPC or C12E8or Triton X-100).During the solubilization,the optimal detergent concentrations were used of20mM DHPC or10mM C12E8or10mM Triton X-100on the basis of the solubilization data obtained from the present study.Protein concentration was kept at4.0–4.5mg/ml and loaded on to a Reactive Red120-agarose column(1.5Â10cm)equilibrated with buffer A containing either of the detergents ata flow rate of6ml/h[44].Proteins were next eluted with step gradient of NaCl(0.5and2M)in buffer A containing the final concentration of the detergents,with the flow rate of12ml/h.Proteins were monitored spectrophotometrically at280nm and assayed for Ca2+-ATPase activity.The enzyme was eluted with0.5M NaCl.The fractions with maximum Ca2+-ATPase activity from several runs were pooled and concentrated by Amicon ultrafiltration cell with YM10membrane(Mr cut off=10kDa).The concentrated pool was adjusted to contain100mM KCl and400A g/ml phosphatidylcholine.2.2.4.3.Sephadex G-100chromatography.The concentrated material (2ml)obtained after Reactive Red120-agarose chromatography was loaded on a Sephadex G-100column(2.3Â92.5cm)equilibrated with buffer A containing100mM KCl with the final concentration of detergents 20mM DHPC,10mM C12E8and10mM Triton X-100and eluted at a flow rate of9ml/h.Proteins were monitored spectrophotometrically at280 nm.All fractions were assayed for Ca2+-ATPase activity and supplemented with400A g/ml phosphatidylcholine.Fractions with highest Ca2+-ATPase activity from several runs were pooled and concentrated by Amicon ultrafiltration cell with YM10membrane(Mr cut off=10kDa).The pooled concentrated fractions with Ca2+-ATPase activity were stored under liquid nitrogen.2.2.5.SDS-polyacrylamide gel electrophoresisSodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli[45]using a minigel Protean II apparatus(Bio-Rad,USA).The protein bands were visualized by silver staining methods[46].2.2.6.Identification of purified Ca2+-ATPase by Western immunoblotCa2+-ATPase purified from the smooth muscle microsomal suspension was identified by western immunoblot,performed by following the method of Towbin et al.[47]using the polyclonal anti-SERCA2b as the primary antibody [48].Horseradish peroxidase conjugated goat anti-rabbit IgG was used as the secondary antibody.The nitrocellulose membrane was developed with4-chloro-1-naphthol(0.2mM).2.2.7.Reconstitution of DHPC,C12E8and Triton X-100purified Ca2+-ATPase into DOPCCa2+-ATPase was reconstituted into the exogenous lipid DOPC by using a combination of methods from the literature[22,24]with some modifications. Briefly,50mg of DOPC in2ml of buffer(50mM MOPS,0.25M sucrose,1 M KCl,1mM MgCl2,0.1mM CaCl2,1mM DTT,0.025%sodium azide;pH 7.5)containing either of the detergents to give a final lipid:detergent ratio of 1:1.5(w/w)[35]was vortex-mixed vigorously for approximately60s to disperse the lipid and left at room temperature(23-C)for90min.The suspension was clarified by sonication for1À2min with a Soni Prep model 150sonifier/cell disrupter.Purified Ca2+-ATPase was solubilized at a ratio of 0.6:1(w/w;detergent:protein)[35]by vortexing for60s and left at room temperature(23-C)for10min followed by further incubation on ice for an additional90min.The solubilized Ca2+-ATPase was centrifuged at low speed to remove insoluble material and aggregates.The clear sample was then mixed with pre-solubilized exogenous lipid to a desired molar ratio of lipid to Ca2+-ATPase(1500:1)[35],and vortex-mixed for30s and left at room temperature for15min.This mixture was incubated at4-C for90min. At the end of the incubation,the detergent was removed by adsorption on Bio-Beads SM-2in two batches of700mg for1h at4-C.The reconstituted vesicles were separated from the Bio-Beads by low-speed centrifugation.The reconstituted proteoliposomes were purified on a discontinuous sucrose gradient.2.2.8.Assay of Ca2+-ATPase activityCa2+-ATPase activity was determined colorimetrically by measuring Ca2+ dependent release of Pi[2].2.2.9.Simultaneous measurement of Ca2+uptake and ATP hydrolysisSimultaneous measurements of Ca2+uptake and ATP hydrolysis were performed with Fura-Red absorbance continuous spectrophotometric assay as described by Karon et al.[49].All assays were performed at790nM.Ionized free calcium was determined from the equation described by Karon et al.[49] with¨50A g of microsomal protein or¨7A g of reconstituted Ca2+-ATPase preparations with the detergents.2.2.10.Characterization of proteoliposomesThe detergent purified Ca2+-ATPase proteoliposomes were treated with protonophore FCCP(0.25A M)as described by Levy et al.[50]and the Ca2+ uptake studies were performed in presence and absence of the protonophore, FCCP by following the method of Karon et al.[49].2.2.11.Fluorescence spectroscopyFluorescence measurements were made by diluting an aliquot of native microsomes(¨50A g/ml of protein)or purified enzyme with the detergents(¨7 A g protein)into2.5ml of buffer{50mM MOPS,2mM EGTA,pH7.5}.The intrinsic fluorescence of tryptophan was measured with a Perkin Elmer spectrofluorimeter model LS50B.Fluorescence spectra were measured with k ex=295nm and k em=340nm and band widths of4and16nm for excitation and emission,respectively.Fluorescence emission spectra of the E1,E1*Ca and E.Mg states of Ca2+-ATPase in native microsomes and in the purified enzyme preparations were recorded at room temperature(23-C).Ca2+and Mg2+ transitions were induced by adding aliquots of1M of CaCl2and5M of MgCl2 stock solutions to give final concentrations of1mM CaCl2and10mM MgCl2, respectively.The sequential additions were followed at a constant emission wavelength of340nm.2.2.12.Circular dichroismCD measurements were made at room temperature with a Jasco J600 spectropolarimeter with a band width of2nm and a scan speed of2nm/min. Each sample was scanned four times;the scans were automatically averaged. The possible effects of light scattering in the CD measurements were minimized by using a CD cell with a narrow path length(0.1cm)and by using identical concentrations of protein and other experimental parameters for each sample in these comparative studies.2.2.13.Quasi-elastic light scatteringQuasi-elastic light scattering was performed with the Nicomp particle sizer from the Pacific Scientific(Silver Spring,MD,USA)with temperature controlled Peltier heating and cooling as described previously[51]to determine the vesicle size of the native microsomes and the other reconstituted proteoliposomes.2.2.14.Statistical analysisData were analyzed by unpaired t test and analysis of variance, followed by the test of least significant differences for comparisonA.Mandal et al./Biochimica et Biophysica Acta1760(2006)20–31 22within and between the groups.P <0.05was considered as significant [52].3.Results and discussion3.1.Characterization of microsomesWe characterized the microsomal fraction at different steps in the preparation process by measuring the activities of cytochrome c oxidase (a mitochondrial marker)[53],acid phosphatase (a lysosomal marker)[54],rotenone insensitive NADPH-cytochrome c reductase (a microsomal marker)[55]and 5V -nucleotidase (a plasma membrane marker)[54].Microsomal fraction showed,respectively,37-fold decrease in sp.activity of cytochrome c oxidase and 26-fold decrease in the sp.activity of acid phosphatase activity compared with 600–15,000Âg pellet.It also showed 45-fold decrease in sp.activity of 5V -nucleotidase,compared with plasma membrane fraction.Furthermore,the microsomal fraction showed,respec-tively,14-fold and 22-fold increase in the sp.activity of rotenone insensitive NADPH-cytochrome c reductase,com-pared with the 600–15,000Âg pellet and the plasma membrane fraction (Table 1).parison of solubilization of microsomes by DHPC,C 12E 8and Triton X-100Solubilization of membrane proteins by suitable detergents with preservation of native structure and function is important for its detailed biochemical and biophysical studies.The purpose of our present research was to investigate the efficacy of the detergents:Triton X-100,C 12E 8and the short chain phospholipid DHPC to solubilize the microsomes isolated from bovine pulmonary artery smooth muscle tissue for structural and functional studies of Ca 2+-ATPase.DHPC is structurally a phospholipid but its short fatty acid chains of seven carbon atoms endow it with detergent like properties [30].It forms micelles [56]rather than bilayers when dispersed in water with a relatively high c.m.c.of 1.4mM and shows a broad size distribution dependent on its concentration and on the NaCl concentration of the suspension [57].DHPC has the advantage that it is available in pure form,it has no net charge,it is stable over a wide pH range of 4–10,and it does not interfere with spectrophotometric measurements [30,35].Microsomes isolated from bovine pulmonary artery smooth muscle tissue were solubilized with DHPC,C 12E 8and Triton X-100at several concentrations,each ranging from 0to 50Table 1Specific activities of different marker enzymes at different steps in the preparation of microsomes from bovine pulmonary artery smooth muscle tissue FractionCytochrome c oxidase Acidphosphatase Rotenone insensitive NADPH-cytochrome c reductase 5V -Nucleotidase600–15,000g pellet 3.34T 0.21 3.40T 0.230.16T 0.020.18T 0.0315,000–100,000g pellet 0.24T 0.04(7)0.33T 0.05(10) 1.68T 0.12(1050) 2.25T 0.16(1250)Microsomes0.09T 0.01(3)0.13T 0.02(4) 2.20T 0.16(1375)0.07T 0.01(39)Plasma membrane 0.08T 0.01(2)0.14T 0.02(4)0.10T 0.01(63)3.12T 0.20(1733)Cytochrome c oxidase activity is expressed as A mol of cytochrome c utilized/30min/mg protein,acid phosphatase activity as A mol of p -nitrophenol/30min/mg protein,NADPH cytochrome c reductase activity as reduction of absorbance of cytochrome c at 550nm/30min/mg protein and 5V -nucleotidase activity as A mol of P i /30min/mg protein.Results are mean T SE (n =4).Values in the parentheses indicate the activity as percentage of that of the 600–15,000g pellet (values of 600–15,000g pellet are set at100%).Fig.1.Ca 2+-ATPase activity of the supernatant obtained after solubilization of microsomes with several concentrations (0–50mM)of DHPC,C 12E 8and Triton X-100and expressed as percentage of starting microsomal Ca 2+-ATPase activity.Total activity in the supernatant:(),DHPC;(),C 12E 8;(),Triton X-100at various detergent concentration as percentage of starting microsomal Ca 2+-ATPase activity.A.Mandal et al./Biochimica et Biophysica Acta 1760(2006)20–3123mM.To compare the microsome-solubilizing ability of these detergents and Ca 2+-ATPase activity,a percentage of the starting amount of native microsomes was determined in the supernatant of the solubilized membranes after pelleting.The supernatant and the resuspended pelleted materials were treated with Bio-Beads SM-2to remove the detergents present prior to being assayed for the enzyme activity.Phosphatidylcholine (400A g/ml)was added to the resulting sample preparation finally in order to avoid aggregation and denaturation of the enzyme in the assay mixture.Fig.1shows the Ca 2+-ATPase activity recovered from the supernatant of the respective detergent-treated samples.In Fig.1,it appears that significantly more activity is observed upon solubilization by the DHPC over a broad range of DHPC concentration than by the C 12E 8and Triton X-100.Fig.2shows the activity associated with the pelleted fractions of each detergent-treated sample.The sp.activities of these preparations in the soluble fractions are shown in Fig.3as a function of detergent concentration.At very low detergent concentration (5mM),the C 12E 8and Triton X-100solubilized enzyme had a higher sp.activity than that solubilized by DHPC.Above 5mM detergent,however,the sp.activity of the DHPC solubilized Ca 2+-ATPase was significantly greater than that solubilized by any of the other two detergents.Additionally,the C 12E 8and Triton X-100solubilized enzyme rapidly lost theiractivitiesFig.2.Ca 2+-ATPase activity of the pellets obtained after solubilization of microsomes with several concentrations (0–50mM)of DHPC,C 12E 8and Triton X-100and expressed as percentage of starting microsomal Ca 2+-ATPase activity.Total activity in the pellets:(),DHPC;(),C 12E 8;(),Triton X-100atvarious detergent concentration as percentage of starting microsomal Ca 2+-ATPaseactivity.),C 12E 8()and Triton X-100().A.Mandal et al./Biochimica et Biophysica Acta 1760(2006)20–3124with increasing detergent concentration and the optimum range for these detergents is,therefore,very narrow (Fig.3).In contrast,the optimum detergent concentration range for DHPC is relatively broad (Fig.3).The above data for DHPC and C 12E 8support the findings of Shivanna and Rowe [35].It appears that the maximum solubilization as well as the Ca 2+-ATPase activity of the microsomes were obtained with 20mM DHPC,10mM C 12E 8and 10mM Triton X-100(Figs.1–3).3.3.Purification of detergent-solubilized Ca 2+-ATPase The detergent-solubilized preparations were purified by Reactive Red 120-agarose chromatography followed by Sephadex G-100gel filtration.The optimum detergent con-centrations for solubilization of the microsomes were chosen as 20mM for DHPC,10mM for C 12E 8and 10mM for Triton X-100(Figs.1–3).The Ca 2+-ATPase activity-containing frac-tions of the Reactive Red 120-agarose column obtained with 0.5M NaCl were pulled,concentrated and loaded on a Sephadex G-100gel filtration column.The ATPase activity containing fractions of the Sephadex G-100chromatography were eluted between 100and 160ml.The Sephadex G-100purified Ca 2+-ATPase showed a single band ¨115kDa (Fig.4A–C).Furthermore,the immunoblot profile of the purified Ca 2+-ATPase with the detergents indicate that the enzyme migrates with a single band at an apparent molecular mass of ¨115kDa (Fig.4D).The sp.activity of the purified enzyme from the DHPC solubilization was significantly greater than that obtained from the preparation solubilized with C 12E 8and Triton X-100(Table 2).The results of the purification scheme are summarized in Table 2.The sp.activity of the DHPC purified Ca 2+-ATPase is 1.9times greater than that obtained from C 12E 8and 2.2times greater than that obtained with Triton X-100solubilized preparations.These data demonstrate that the DHPC solubili-zation results in a significantly greater quantity of high sp.activity of Ca 2+-ATPase than the other two detergents.The present comparative studies of the result from solubilization and purification of microsomal Ca 2+-ATPase demonstrate the inherent variations exhibited by various detergent groups when interacting with the microsomes.The three detergents employed in this study:DHPC,C 12E 8and Triton X-100represent well-differentiated groups of soluble amphiphiles,i.e.,the phospholipid and non-ionic detergents.The DHPC solubilization consistently provides greater yields,with specific activities significantly greater over a wide concentration range of the detergent than that achieved either by C 12E 8or the Triton X-100(Table 2and Fig.3).The yield of total units of activity at the optimal solubilization concentration for DHPC is 32%and 56%higher than that obtained with C 12E 8and Triton X-100,respectively (Fig.1).CombiningtheFig.4.(A)7.5%SDS-PAGE profile of Ca 2+-ATPase purification with ne 1,microsome;lane 2,DHPC solubilized microsome;lane 3,Reactive Red 120-agarose flow through;lane 4,0.5M NaCl eluate of Reactive Red 120-agarose;lane 5,Sephadex G-100eluate;lane 6,standard mol.wt.markers.(B)7.5%SDS-PAGE profile of Ca 2+-ATPase purification with C 12E ne 1,microsome;lane 2,C 12E 8solubilized microsome;lane 3,Reactive Red 120-agarose flow through;lane 4,0.5M NaCl eluate of Reactive Red 120-agarose;lane 5,Sephadex G-100eluate;lane 6,standard mol.wt.markers.(C)7.5%SDS-PAGE profile of Ca 2+-ATPase purification with Triton ne 1,microsome;lane 2,Triton X-100solubilized microsome;lane 3,Reactive Red 120-agarose flow through;lane 4,0.5M NaCl eluate of Reactive Red 120-agarose;lane 5,Sephadex G-100eluate;lane 6,standard mol.wt.markers.(D)Western blot profile of purified Ca 2+ne 1,control;lane 2,DHPC purified Ca 2+-ATPase;lane 3,C 12E 8purified Ca 2+-ATPase;lane 4,Triton X-100purified Ca 2+-ATPase;lane 5,standard mol.wt.markers.Table 2Purification of Ca 2+-ATPase from bovine pulmonary artery smooth muscle microsomes Purification stepsDetergents used Total protein (mg)Total activity (Units a )Specific activity (Units/mg)Foldpurification Recovery %Microsomes–50745.600.09–100Detergent solubilized microsomesDHPC 53.8830.550.567667C 12E 847.5520.970.441546Triton X-10042.4610.690.252323Reactive Red 120-agarose chromatography DHPC 3.4918.56 5.3185941C 12E 82.969.073.0643420Triton X-100 2.637.31 2.7793116Sephadex G-100chromatographyDHPC 1.38212.378.9519927C 12E 81.320 6.21 4.7045214Triton X-1001.2695.174.0744511aUnit=A mol Pi/min at 37-C.A.Mandal et al./Biochimica et Biophysica Acta 1760(2006)20–3125。